Spermatozoa acquire their motility and fertilization capacity as they traverse the epididymis. The lumen of the epididymis is acidic and has a low bicarbonate concentration: both factors are important for keeping sperm quiescent during their maturation and storage. In the previous funding periods, we showed that luminal acidification is achieved by clear cells, via the proton pumping V-ATPase, and that it can be regulated via crosstalk with surrounding epithelial cells. We now plan to dissect the intercellular communication networks between principal cells and clear cells using a systems biology approach that links molecular events in signaling pathways to cellular events in the intact epididymis in vivo. We propose that stimulation of principal cells by basolateral paracrine factors during sexual arousal leads to subsequent activation of clear cells via ATP and bicarbonate that are secreted into the epididymal lumen. This would allow luminal pH to be restored to its resting acidic value. Alternatively, some of these factors might directly stimulate clear cells to secrete protons. In Aim 1A, multiphoton intravital microscopy (IVM), functional ion flux assays of proton secretion and intracellular calcium measurements will be used to examine clear cell activation following stimulation of principal cells by basolateral hormones and agonists. These effects will be compared with those elicited by luminal ATP, adenosine and bicarbonate. Aim 1B will identify and characterize the apical purinergic receptors (e.g. A2B, P2X4), and bicarbonate transporters (e.g. NBC3) involved in the response to luminal mediators (ATP, adenosine and bicarbonate), using LC-MS/MS, RT-PCR, in situ hybridization, western blotting and immunofluorescence. Their role in clear cell activation will be analyzed, as described in Aim 1A. Aim 2 will monitor luminal ATP and bicarbonate secretion by principal cells by IVM following basolateral stimulation, using a novel luciferase assay and BCECF-dextran, respectively. The role of critical apical transporters and regulators involved in ATP (e.g. ClC-3, CFTR) and bicarbonate (e.g. SLC26A6) secretion will be examined. The role of CFTR, which plays a critical role in male fertility, will be further dissected in immortalized epididymal cell lines, and the consequences of cystic fibrosis-associated CFTR mutations will be determined. Aim 3 will explore the direct activation of clear cells by hormones and agonists, without principal cell involvement. Cell morphology, V-ATPase apical recruitment, V-ATPase-dependent proton secretion and intracellular calcium will be examined in EGFP-expressing clear cells isolated by FACS from our unique transgenic mice. These studies will provide new insights into the mechanisms by which epithelial cells work in a concerted manner to establish and maintain an acidic luminal environment that is permissive for sperm maturation and storage in a quiescent state. In summary, we propose to use a multidisciplinary approach to address a problem that is central to our understanding of post-testicular regulation of male fertility. Data generated here will provide novel insights into the male reproductive tract in particular, as well as the physiology of acid/base transporting epithelia in general.